The extracellular matrix is that material which surrounds the muscle and is the major component of connective tissue of all mammals. The extracellular matrix provides for structural integrity, and promotes cell migration and cellular differentiation. As part of these functions, the extracellular matrix has been shown to support adhesion for various types of cells in vitro. Molecules such as the collagens, fibronectin, vitronectin, laminin, von Willebrand factor, thrombospondin, bone sialoprotein, fibrinogen, and tenacin have been found to possess this property of mediating cell adhesion.
The above cell-adhesive molecules have been found to exhibit a structural similarity in their respective binding sites, each of which contains the amino acid sequence arginine-glycine-aspartic acid, or RGD using single letter nomenclature (infra). The cell-binding site in fibronectin has been reproduced synthetically. In turn, the cellular receptor site for fibronectin has been identified for various cells. In addition, cellular receptors that recognize RGD-containing sequences in other extracellular matrix proteins (e.g., the vitronectin receptor) have been identified.
Such cellular receptors, responsive to RGD-containing proteinaceous compounds, have been characterized. The complete, primary structure of the fibronectin receptor has been deduced from cDNA, and physical properties have been determined. Argraves, et J.Biol. Chem. 261: 12922 (1986); Argraves J.Cell Biol. 105: 1183 (1987). The protein exists at the cell surface as a heterodimeric complex (although the larger polypeptide is enzymatically processed) having both polypeptide chains inserted into the membrane. Each chain extends 30-40 residues into the cytoplasmic space, and at least one of the cytoplasmic peptides appears to interact with the cytoskeleton. Horwitz et al., Nature 320: 531 (1986). The larger of the two polypeptides, the .alpha. subunit, contains a number of regions that are structurally similar to calmodulin and that apparently mediate the binding of calcium to the receptor. The presence of such divalent cations is required for the receptor to bind ligand. The .beta. subunit is somewhat smaller and conformationally compact due to numerous intrachain disulfide bonds. The cytoplasmic domain of the .beta. subunit contains a potentially phosphorylated tyrosine. Hirst et al., PNAS-USA 83: 6470 (1986 ); Tamkun et al., Cell 46: 271-282 (1986).
Other RGD-directed receptors, as well as other "orphan" receptors the ligand for which is unknown, have also been characterized. This putative RGD commonality of the ligand matrix proteins has revealed a superfamily of cell surface receptor proteins that share a high degree of structural similarity and probably also functional similarity. The members of this superfamily of cell surface proteins collectively are known as the integrins. The integrins can be grouped on the basis of the identity of their .beta. subunit. The .beta. subunit, as disclosed above for the fibronectin receptor, is compact due to a high degree of cross-linking. The first group of integrins includes the very late activation antigen (VLA) proteins, which themselves include the fibronectin receptor (VLA-5), the collagen receptor (VLA-2), and the laminin receptor. The second group includes the lymphocyte associated antigen-1 (LFA-1), macrophage antigen-1 (MAC-1), and p150,95. The third group includes the vitronectin receptor, and platelet glycoprotein gpIIb/IIIa. Hynes, Cell 48: 549 (1987); Hemler, Immunol. Today 9: 109 (1988); Springer et al., Annu. Rev. Immunol. 223 (1987); Kishimoto et al., Leukocyte Integrins, in: Leukocyte Adhesion Molecules, T. A. Springer, D. C. Anderson, A. S. Rosenthal, and R. Rothlein, Eds., Springer-Verlag, New York, pp. 7-43 (1989).
The RGD-directed receptor present on platelets that binds fibronectin, vitronectin, fibrinogen, and von Willebrand factor has also been purified. This receptor is the gpIIb/IIIa protein complex. This receptor is thus not specific to one extracellular matrix protein, as are the above fibronectin and vitronectin receptors. It has been proposed that this lack of specificity is correlated to the lack of conformational specificity in the ligands. Other work has suggested that specificity can be achieved with relatively short, conformationally restricted synthetic peptides containing the RGD sequence. For a literature summary, see: Pierschbacher et al., Nature 309: 30 (1984); Pierschbacher et al., pNAS-USA 81: 5985 (1984); Ruoslahti et al., Cell 44: 517 (1986); Pierschbacher et al., Science 238: 491 (1987); Pierschbacher et al., J.Biol.Chem. 262: 17294 (1987); Hynes, Cell 48: 549 (1987); Ruoslahti, Ann. Rev. Biochem. 57: 375 (1988). It has also been proposed that the receptor affinity for its peptide ligand may be altered as the stereoconformation, or three-dimensional shape, of the peptide is restricted, typically by cyclization. Pierschbacher and Ruoslahti, PCT International Publication WO 89/05150 (1989). However, the publication states that the cyclic peptide of the invention (see FIG. 2 and Example V) was ineffective in inhibiting attachment to fibronectin.
A limited number of compounds containing sequences of natural amino acids or derivatives other than RGD may also possess the capability for affecting cell adhesion. These non-RGD-containing peptides are not well characterized. See. Graf, J. et al., Cell 48:989 (1987); Kloezewiak, M. et al,, Biochemistry 23:1767-1774 (1984); Wayner, E.A., et al., J. Cell. Biol. 109:1321 (1989).
All publications, patents and other reference materials to which reference is made in the present specification are incorporated herein by reference.